Enzymes of Heavy-Metal-Resistant and Non-Resistant Populations of Silene cucubalus and Their Interaction with Some Heavy Metals in vitro and in vivo

The in vitro and in vivo effects of copper, zinc, cadmium, nickel, cobalt, and manganese on nitrate reductase, malate dehydro-genase, isocitrate dehydrogenase, and glucose-6-phosphate dehydrogenase of zinc-, copper- and non-resistant populations of Silene cucubalus were investigated. During the in vitro experiments no resistant enzyme could be detected; enzymes of resistant and non-resistant ecotypes had a similar sensibility to all the metals. Nitrate reductase was the most sensitive enzyme. During the in vivo experiments remarkable differences were found. The nitrate reductase and the isocitrate dehydrogenase of the zinc-resistant population were activated when adding zinc to the culture medium, especially the nitrate reductase showed high activities at zinc concentrations where the nitrate reductase of the non-zinc-resistant populations was nearly completely inhibited. The zinc-resistant ecotype had a real need for zinc.     

Zinc Phosphate Transformations by the Paxillus involutus/Pine Ectomycorrhizal Association

In this research, we investigate zinc phosphate transformations by Paxillus involutus/pine ectomycorrhizas using zinc-resistant and zinc-sensitive strains of the ectomycorrhizal fungus under high- and low-phosphorus conditions to further understand fungal roles in the transformation of toxic metal minerals in the mycorrhizosphere. Mesocosm experiments with ectomycorrhizas were performed under sterile conditions with zinc phosphate localized in cellophane bags: zinc and phosphorus mobilization and uptake by the ectomycorrhizal biomass were analyzed. In the presence of a phosphorus source, an ectomycorrhizal association with a zinc-resistant strain accumulated the least zinc compared to a zinc-sensitive ectomycorrhizal association and non-mycorrhizal plants. Under low-phosphorus conditions, mycorrhizal seedlings infected with the zinc-resistant strain increased the dissolution of zinc phosphate and zinc accumulation by the plant. Extended X-ray absorption fine structure analysis of both mycorrhizal and nonmycorrhizal roots showed octahedral coordination of zinc by oxygen-containing ligands such as carboxylates or phosphate. We conclude that zinc phosphate solubilization and zinc and phosphorus uptake by the association depend on ectomycorrhizal infection, strain of the mycobiont, and the phosphorus status of the matrix.     

Der Einfluss der Phosphatversorgung sowie die Wirkung von ionogenem und chelatisiertem Zink auf die Zink- und Phosphataufnahme einiger Schwermetallpflanzen

In physiologischen Untersuchungen an Schwermetallpflanzen wurde die hohe Zinkverträglichkeit für Silene cucubalus var. humilis bestätigt und für Thlaspi alpestre ssp. calaminare neu festgestellt. In Wasserkulturversuchen mit Silene cucubalus var. humilis wirkte Zinksulfat bereits bei einer Gabe von 100 mg Zn/1 im Verlauf einer Vegetationsperiode toxisch. Hingegen wurde Zink-Chelat noch bei einer Konzentration von 600 mg Zn/I ertragen. Durch Chelatgaben kann demnach die Zinkverträglichkeit der Schwermetallpflanzen wesentlich erhöht werden. Die untersuchten Schwermetallpflanzen Silene cucubalus var. humilis, Armeria maritima ssp. halleri und Cardaminopsis halleri nahmen wesentlich mehr Zink aus einer zinksulfathaltigen als aus einer zinkchelathaltigen Nährlösung auf. Dabei dürfte die toxische Grenze durch die in den Sproß gelangte Zinkmenge bestimmt werden. Die Toxizitat scheint dann erreicht zu sein, wennsich bei hoher Gesamtaufnahme über 80% des Zinks im Sproß befinden. Zwischen dem Zink- und dem Phosphatgehalt der Sprosse ergab sich in den Zink-sulfatreihen eine negative Korrelation, die jedoch für die Zink-Chelatreihen nicht bestatigt werden konnte.     

Ethylene enhances gibberellin levels and petiole sensitivity in flooding-tolerant Rumex palustris but not in flooding-intolerant R. acetosa

The role of gibberellin (GA) and ethylene in submergence-induced petiole elongation was studied in two species of the genus Rumex. Analysis of endogenous GAs in the flooding-tolerant Rumex palustris Sm. and the intolerant Rumex acetosa L. by gas chromatography-mass spectrometry showed for both species the presence of GA₁, GA₄, GA₉, GA₁₉, GA₂₀ and GA₅₃. Gas chromatography-mass spectrometry analysis of R. palustris petiole tissue of submerged plants showed an increase in levels of 13-OH GAs, especially GA₁, compared with drained plants. This effect could be mimicked by application of 5 μL L⁻¹ ethylene. In R. acetosa, no differences between levels of GAs in drained or submerged plants were found. In R. palustris, both submergence and ethylene treatment sensitized petioles to exogenous gibberellic acid (GA₃). In R. acetosa the effect was opposite, i.e. submergence and ethylene de-sensitized petioles to GA₃. Our results demonstrate the dual effect of ethylene in the submergence response related to flooding tolerance, i.e. in the flooding-tolerant R. palustris ethylene causes an increased concentration of and sensitivity to GA with respect to petiole elongation while in the intolerant R. acetosa ethylene reduces growth independent of GAs. Received: 5 November 1996 / Accepted: 8 February 1997     

Altitude-related changes in activities of carbon metabolism enzymes in <Emphasis Type="Italic">Rumex nepalensis</Emphasis>

Activities of some enzymes related to carbon metabolism were studied in different ecotypes of Rumex nepalensis growing at 1 300, 2 250, and 3 250 m above mean sea level. Activities of ribulose-1,5-bisphosphate carboxylase/oxygenase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, and glutamine synthetase increased with altitude, whereas activities of malate dehydrogenase, NAD-malic enzyme, and citrate synthase did not show a significant difference with change in altitude.     

Phytoextraction of Zinc: Physiological and Molecular Mechanism

Zinc is an essential trace element, necessary for plants, animals, and microorganisms. Zn is required for many enzymes as a catalytic cofactor, for photosynthetic CO₂ fixation, and in maintaining the integrity of bio-membranes. However, Zn is potentially toxic when accumulated beyond cellular needs. Phytoextraction technique, which is a part of phytoremediation, has opened new avenues for remediation of Zn-contaminated places. Hyperaccumulators like Thlaspi caerulescens and Arabidopsis halleri have been identified, which can accumulate up to 40,000 mg kg^?1 Zn in the aerial parts of the plant body. Carboxylic acids, primarily malate, citrate, and oxalate, and amino acids are found to play an important role in Zn hyperaccumulation. Transmembrane metal transporters are assumed to play a key role in Zn metal uptake, xylem loading, and vacuolar sequestration. Members of CDF (cation diffusion facilitator) and ZIP (zinc-regulated transporter, iron-regulated transporter like protein) family have been implicated in Zn-metal-tolerance mechanisms. A potential metal-binding motif, containing multiple histidine residues, is found in the variable regions of almost all of the ZIP family, including ZIP1, ZIP2, ZIP4, ZRT1, and ZRT2. Overexpression of some Zn metal transporter genes like TcZNT1 (Thlaspi caerulescens Zn transporter1), TcHMA4 (Thlaspi caerulescens heavy metal ATPase) in Thlaspi caerulescens, AhMTP1;3 (Arabidopsis halleri metal transporter1;3) in Arabidopsis halleri, and PtdMTP1(Poplar metal transporter1) from a hybrid poplar confer Zn hypertolerance in Thlaspi, Arabidopsis, and Poplar plant species.     

Soil seed banks in a heavy-metal polluted grassland at Prayon (Belgium)

Seed banks were investigated in a grassland dominated byAgrostis capillaris and Thlaspicaerulescens, established on soils polluted by atmospheric depositsof SO₂ and heavy-metal rich dust (Zn, Pb, Cd) from an ore-smelter inPrayon (E Belgium). A total of 155 soil cores (0–7 depth)distributed among 10 plots were sampled in November and March. Each samplingplot was characterised for the botanical composition and standing biomass of theestablished vegetation as well as for four environmental parameters (soil pH andextractable zinc, volume percentage of stones, relative altitude within thesite). Very high densities of germinable seeds were found (grand mean ±SE, pooled over dates and plots: 28,950 ± 6830 ). Agrostis capillaris represented 95% of theseeds, followed by Thlaspi caerulescens (3%); the remaining2% were shared among 24 other species. Two thirds of the seeds were concentratedin the upper 3 of the soil. The high seed density might be dueto a low rate of microbial decay of seeds due to heavy metal toxicity. The seedbank is almost entirely derived from those few heavy-metal tolerant species thathave re-colonised the polluted area in the last three decades. Only two species(Anagallis arvensis and Urtica dioica)not represented in the established vegetation and with seeds concentrated in thelower soil layer are probably derived from seed buried prior to the onset ofheavy-metal pollution. This study provides the first data suggesting that theheavy-metal hyperaccumulator Thlaspi caerulescens possessespersistent seed banks. Seed densities of Agrostiscapillaris, Thlaspi caerulescens andRumex acetosa were positively correlated with the standingbiomass of grasses and negatively correlated with the volume percentage occupiedby stones in the soil. It is argued that small-scale variation in microclimaticfactors that cue germination (fluctuations in soil temperature and moisture) canaccount for the observed pattern of spatial variation in seed density. From thestandpoint of restoration management, the results indicate that little change inthe floristic composition of the heavy-metal tolerant grasslands of Prayon canbe expected from the recruitment of buried seeds.

     

Interactions between Apion species (Coleoptera: Curculionidae) and Polygonaceae. I. Apion curtirostre Germ, and Rumex acetosa L.

Abstract.

• 1 Apion curtirostre is the commonest weevil attacking Rumex acetosa in the Lancaster area.
• 2 The ovipositional behaviour, larval development, emergence and hibernation of A. curtirostre are described.
• 3 From dissection of samples of stems of R. acetosa, abundance curves for the stages of A. curtirostre and its chalcid parasitoids were constructed and the between-stage mortalities determined.
• 4 There is an exponential relationship between R. acetosa stem height and number of A. curtirostre eggs laid.
• 5 Male stems of R. acetosa appear earlier and are smaller than female stems. Taller stems, at least in female plants, live longer than shorter ones.
• 6 Apion survival is lower in male than in female stems.
• 7 Stem sex is the primary determinant of opposition choice with stem thickness a secondary determinant.
• 8 A. curtirostre eggs introduced experimentally into stems of four Rumex species survived significiantly better in R. acetosa than in any other species. Survival in R. acetosa female plants was twice that in males.
• 9 Survival of A. curtirostre eggs at densities of 4, 8 and 16 per R. acetosa stem was twice that at densities of 32 and 64 per stem.
• 10 The stem size of female R. acetosa was reduced as the number of attacking weevils increased. There was no significant effect in males. No adverse effect on seed production was detected.
• 11 The nature of the relationship between A. curtirostre and R. acetosa is discussed.

     

Seed survival and seasonal emergence in some species of Geranium, Ranunculus and Rumex

Seeds of 14 species were collected, usually in each of 2 or 3 yr, and mixed with the top 7.5 cm of sterilised soil confined in cylinders sunk in the ground outdoors and cultivated three times yearly. Seedling emergence was recorded for at least 3 and usually 5 yr. Most seedlings of Ranunculus bulbosus and Rumex acetosa appeared during the autumn of sowing, while the main emergence of Geranium pratense. Ranunculus acris, Rumex conglomeratus and R. maritimus was in the following spring. Emergence of G. pyrenaicum and G. robertianum took place throughout much of the year after sowing. Seeds of all these species were relatively short-lived in cultivated soil, and few seedlings appeared after the second year. Seed survival was greater in G. dissectum. G. molle, Rumex acetosella. Ranunculus sceleratus and especially R. repens, of which emergence in the fifth year after sowing amounted to 4% of the seeds sown. Emergence from the persistent seed bank was mainly from May to September for G. dissectum and G. molle and during autumn for Ranunculus flammula and R. sceleratus, while seedlings of Ranunculus repens and Rumex acetosella appeared sporadically over most of the year.     

In vitro organogenesis in two dioecious species,Rumex acetosella L. and R. acetosa L. (Polygonaceae)

Callus cultures were established from dioecious plant species Rumex acetosella and R. acetosa, using cotyledons, hypocotyls and stem tips of aseptically germinated seedlings as primary explants. Cultures were also established from male and female R. acetosella adult plants, starting from vegetative lateral buds. Cell division was induced using a high 2,4-D concentration, while bud induction and multiplication were stimulated on a medium with high BAP/IAA ratio. Cotyledon fragments of both species produced only rhizogenic calli. Hypocotyl-derived calli of R. acetosella produced buds, while those of R. acetosa showed no bud forming response under these conditions. Bud multiplication occurred in stem tip cultures of both species and in lateral bud cultures of R. acetosella. Calli derived from male plants produced more buds than those from female. Shoots were easily rooted using IBA, and plantlets were effectively transferred to soil. Flowering was not induced in culture. The sex of regenerated male and female plants was not altered by the culture conditions.     

Isolation of RNA from floral tissue ofRumex acetosa (Sorrel)

Flower tissue ofRumex acetosa was previously intractable for the isolation of RNA using standard methods, due probably to its high level of polysaccharides. Extraction at low pH, precipitation of polysaccharides with potassium acetate followed by precipitation of RNA with lithium chloride yielded high-quality RNA that was suitable for northern hybridisation,in-vitro translation, poly(A)⁺ RNA selection, and subsequent cDNA synthesis.     

Occurrence of Two Pathways for Malate Oxidation in Bacteroids Isolated from Sesbania rostrata Stem Nodules during C(2)H(2) Reduction

Malate oxidation supported C₂H₂ reduction by bacteroids isolated from Sesbania rostrata stem nodules. Optimal activity reached 7.5 nanomoles per minute per milligram of dry weight and was in the same order of magnitude as that observed with succinate but always required a lower O₂ tension. Malate dehydrogenase (EC 1.1.1.37), purified 66-fold from bacteroids, actively oxidized malate (K_m = 0.19 millimolar). Malic enzyme (EC 1.1.1.39) from Sesbania bacteroids had a lower affinity for malate (K_m = 2.32 millimolar). Both enzymes exclusively required NAD⁺ as cofactor and required an alkaline pH for optimal activity. 2-Oxoglutarate and oxalate, inhibiting malate dehydrogenase and malic enzyme, respectively, were used to specifically block each malate oxidation pathway in bacteroids. The predominance of malate dehydrogenase activity to support bacteroid N₂ fixation was demonstrated. The inhibition of O₂ consumption by 2-oxoglutarate confirmed the importance of the malate dehydrogenase pathway in malate oxidation. It is proposed that the utilization of malate, with regard to O₂, is important in a general strategy of this legume to maintain N₂ fixation under O₂ limited conditions.     

Pollen and non-pollen palynomorph evidence of medieval farming activities in southwestern Greenland

Radiocarbon dating, pollen and non-pollen palynomorph analyses from a lake core were used to establish the timing and effects of farming activities around Lake Igaliku, Eastern Settlement, Greenland. The absence of agro-pastoral impact before the medieval colonization by Europeans provides an opportunity to understand the development of farming activity in a pristine landscape. The results show that the first phase of clearance and grazing pressure, without the expansion of the Norse apophyte (native plant, in habitats created by humans) Rumex acetosa type, could have occurred in the 9–10th century a.d. The presence of Norse settlers and livestock is clearly recorded from the 11–12th century a.d. with increasing frequencies of the Norse apophytes Rumex acetosa type and Ranunculus acris type, and coprophilous fungi. This colonization phase is followed by a period of decreasing human impact at the beginning of the 14th century, with a decrease in weeds, apophytes and coprophilous fungi suggesting a reduced grazing pressure. The regrowth of Salix and Betula and the disappearance of anthropogenic indicators except Rumex acetosa type between the 15th and 18th century demonstrate the abandonment of the settlement, until the development of contemporary agriculture in the 20th century.     

Sex ratio variation and spatial segregation of the sexes in populations ofRumex acetosa andR. acetosella (Polygonaceae)

Populations ofRumex acetosa andR. acetosella were studied during two growth seasons. The ramet sex ratios ofR. acetosa were always female-biased. InR. acetosella the sex ratios expressed more variation but were mostly female-biased. In both species the sex ratios commonly varied between subpopulations reflecting a partial spatial segregation of the sexes. No marked differences between sexes in vegetative vigour were detected in either species. Interactions between sex ratios, various soil characteristics and population densities were determined. Possible mechanisms for causing biased sex ratios and partial spatial segregation of the sexes are discussed.     

Chromosome painting of Y chromosomes and isolation of a Y chromosome-specific repetitive sequence in the dioecious plant Rumex acetosa

The dioecious plant Rumex acetosa has a multiple sex chromosome system: XX in female and XY₁Y₂ in male. Both types of Y chromosome were isolated from chromosome spreads of males by manual microdissection, and their chromosomal DNA was amplified using degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR). When the biotin-labeled DOP-PCR product was hybridized with competitor DNA in situ, the fluorescent signal painted the Y chromosomes. A library of Y chromosome DNA was constructed from the DOP-PCR product and screened for DNA sequences specific to the Y chromosome. One Y chromosome-specific DNA sequence was identified and designated RAYSI (R. acetosa Y chromosome-specific sequence I). RAYSI is a tandemly arranged repetitive DNA sequence that maps to the 4’,6-diamidino-2-phenylindole bands of both Y chromosomes. Received: 22 December 1998; in revised form: 22 March 1999 / Accepted: 23 March 1999     

Inability to solubilize phosphate in limestone soils—key factor controlling calcifuge habit of plants

Germination, seedling establishment and growth of calcifuge plants in Swedish limestone soils of Archean and Ordovician age were studied. As previously demonstrated for Viscaria vulgaris, establishment of Rumex acetosella and Silene rupestris did not succeed unless CaHPO₄ (at the rate of 10 mmol dm^-3 of soil) was supplied. Growth of Deschampsia flexuosa was enhanced by phosphate addition, whereas establishment success of Jasione montana was poor, regardless of phosphate treatment. Establishment and growth in an acidic gneiss soil, used as a reference for the species studied, was good. Total, total inorganic, exchangeable, and soil solution P were considered in all soils and treatments. It is proposed that the calcifuge behaviour of plants is quite often caused by inability to solubilize the native phosphate of limestone soils.     

Response to food plant stimuli by larvae of the mustard beetle Phaedon cochleariae

Larvae of the mustard beetle Phaedon cochleariae Fab. are attracted to the cruciferous host plant by yellow and green wavelengths of light and the odour of mustard oils. The mustard oils induce biting, and feeding continues in the presence of mustard oil glucosides in the plant. Leaf toughness and texture then affect food intake and larval growth and survival.

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Zusammenfassung Visuelle und geruchliche Reize sind dafür verantwortlich, dass Larven von Phaedon cochleariae von der Wirtspflanze angezogen werden. Wahrscheinlich ist für die Lokalisierung des Futters hauptsächlich die visuelle Reaktion auf gelbgrüne Farbe von 520–570 nm wichtig. Dies wird ergänzt durch den Geruch der Senföle mit einer Konzentration an der Quelle von 0,25–250 ppm, welcher die Larven zu grösserer Suchaktivität anregt. Senföle veranlassen die Larven zum Beissen eines Substrats. Der Frass hält an, wenn der Geschmacksreiz von Glucosiden vorhanden ist. Frassmenge und Wachstumsgeschwindigkeit werden beeinflusst durch die Zähigkeit der Blätter und eventuell durch die Blatttextur und den Nährstoffgehalt.

     

Life history variation in the heavy metal tolerant plant Thlaspi caerulescens growing in a network of contaminated and noncontaminated sites in southern France: role of gene flow, selection and phenotypic plasticity

* Here we explore life history differences in a set of neighbouring metallicolous and nonmetallicolous populations of the heavy metal tolerant plant Thlaspi caerulescens. * We contrasted data from field observations and from a common garden experiment, in which soil zinc (Zn) concentration and light availability were manipulated, and data on microsatellite molecular variation. * The two ecotypes showed few differences in life history in the field, but large differences in their response to Zn concentration in the common garden. Soil toxicity affected most characters in nonmetallicolous plants, while it had no effect on metallicolous plants. The two ecotypes responded similarly to light. Genetic differentiation for quantitative characters between ecotypes contrasted with the absence of differentiation for microsatellites. Conversely, populations of the same ecotype showed similar responses to Zn, despite their high differentiation for molecular markers. * We conclude that divergent selection related to soil toxicity has had a predominant role in shaping life history differences between ecotypes, gene flow weakly opposing local adaptation despite geographical proximity.     

Carbon Dioxide Efflux from Leaves in Light and Darkness

Efflux of carbon dioxide in light and darkness was measured at low ambient CO₂ concentrations in leaves of Rumex acetosa. Light carbon dioxide production (photo-respiration) was found to depend on irradiance and to differ from dark production as to the response to temperature and ambient concentrations of O₂ and CO₂. These observations support previously made suggestions that photorespiration follows a different metabolic pathway to dark respiration.     

Formation of l(-)malate by Saccharomyces cerevisiae during fermentation

Summary When grown in a synthetic medium most of the 51 strains of the genera Saccharomyces, Saccharomycodes, Zygosaccharomyces and Schizosaccharomyces investigated formed l-malate during fermentation. The quantity varied between 0.1 and 2.6 g malate per liter. Two strains of Saccharomyces cerevisiae synthesized malate at a rate of about 1.5 g/l. Malate was liberated during the growth phase and not metabolized during the stationary phase. Optimum malate formation was observed at a sugar concentration of about 20% (w/v), at pH 5 and at suboptimal nitrogen concentrations of less than 300 mg N/liter. Of the amino acids aspartate and glutamate were most favourable. If ammonium salts were used as the nitrogen source, significant amounts of malate were formed when the pH was kept constant by buffering. Trace metals had no or only little influence on malate synthesis. Biotin and pantothenate were essential for growth. Added ¹⁴CO₂ led to the formation of approximately equal quantities of labelled malate and succinate by S. cerevisiae strain 52, whereas about ten times more malate than succinate was formed by Saccharomyces uvarum. Avidin strongly inhibited the formation of malate while the inhibiton of succinate synthesis and of growth was comparatively much less. Malate is obviously formed by reduction of oxalacetate, the synthesis of which is catalysed by a biotin-dependent pyruvate carboxylase.